PROJECT SUMMARY Ewing sarcoma is a highly aggressive bone and soft tissue cancer that is caused by the EWS-FLI1 fusion protein. The EWS-FLI1 oncoprotein functions, in part, as an aberrant transcription factor and is required for tumor growth and survival. However, directly targeting EWS-FLI1 with drugs has been challenging and, as a result, there is a critical need to identify downstream targets of EWS-FLI1 and unique vulnerabilities incurred by the oncoprotein. In order to identify downstream targets of EWS-FLI1, we used human embryonic stem cells that express inducible EWS-FLI1 to model the initiation and development of Ewing sarcoma in a genetically defined system. We then used this model system and a gene expression based approach to identify that Ewing sarcoma cells are uniquely vulnerable to inhibitors of ribonucleotide reductase (RNR), including gemcitabine, which impair DNA replication by blocking the synthesis of deoxyribonucleotides. Moreover, we have also identified that the inhibition of RNR in Ewing sarcoma cells results replication stress, activation of the unfolded protein response, and a block in the synthesis of proteins required for the response to impaired DNA replication. Notably, the combination of gemcitabine with an inhibitor of checkpoint kinase 1 (CHK1), the major regulator of the response to impaired DNA replication, results in synergy in vitro and a significant prolongation of mouse survival in xenograft experiments. Guided by strong preliminary data, we will now pursue the following specific aims: 1) dissect the molecular basis of the activation and regulation of the unfolded protein response in Ewing sarcoma cells treated with inhibitors of ribonucleotide reductase; 2) test the in vivo efficacy of gemcitabine and a second-generation CHK1 inhibitor, prexasertib, using Ewing sarcoma xenograft models; and 3) use high-throughput screening to test candidate therapeutics identified using our gene expression based approach for activity against Ewing sarcoma cells. These aims will be tested using a combination of approaches in cancer cell lines, cell line xenografts, patient-derived xenografts, and our isogenic, genetically defined cell lines. This work will be significant because it is expected to have translational relevance for the treatment of children and adults with Ewing sarcoma tumors, as well as lead to a broader understanding of the basic mechanisms of tumorigenesis in Ewing sarcoma tumors. The proposed research is innovative because it integrates a novel, stem cell model of Ewing sarcoma with a gene expression signature based screening approach to identify new therapeutic targets.